1 | P a g e

Attachment D - USP Monograph 2012

USP MONOGRAPH Helicoll

Enhancing Life Through Collagen

2 | P a g e

BACKGROUND

1.1 Wound Healing and Collagen:

The wound healing process is a complex series of events that begins at the moment of

injury and can continue for months to years. This process has four phases: the blood

clotting phase, inflammatory phase, the proliferative phase, and the maturational

phase.1

Collagen, the most abundant protein found in the body, is the main supportive protein

of cartilage, connective tissue, tendon, skin, and bone. There are at least 13 different

types of collagen. Types 1, 3, 4, 5, and 7 are specific for skin.2

Collagen plays an integral part during each phase of wound healing and is an excellent

hemostatic agent as it absorbs 40 - 60 times its weight in fluid. Collagen exposed

during wound formation activates the clotting phase, when the collagen is native and

bioactive, and is responsible for cell signalling that influences the migration of

inflammatory cells to the wound bed.1-4

Collagen dressings have been used in various forms for tissue repair and wound

healing5 as it constitutes more than 80% of the structural proteins of the body.

Compared to many other modern non-biological dressings, collagen dressings remain a

poorly understood and probably underused material. Biodegradable (bio utilized)

collagen dressings are derived from animal tissues. These collagen dressings maintain

a physiologically moist microenvironment that promotes healing and the formation of

granulation tissue.6

The healing of skin tissue requires the development of a vascularized granular tissue

bed, filling of large tissue defects by dermal regeneration, and the restoration of a

continuous epidermal keratinocyte layer. Several experimental results suggest that

collagen is an ideal material for tissue regeneration compared to other non-biological

wound healing materials.6-8

In a wound where the basement membrane has been destroyed, similar to a second or

third degree burn, the wound is re-epithelialised from the normal cells in the periphery

and from the skin appendages provided the basement is intact (e.g., hair follicles, sweat

glands). The granulation phase and tissue deposition require nutrients supplied by the

capillaries, and failure for this to occur results in a chronically unhealed wound.

Fibroblasts differentiate and produce ground substance and then collagen. Many

different cytokines are involved in the proliferative phase of wound repair. The steps

and the exact mechanism of control have not been elucidated. Some of the cytokines

include PDGF, insulin-like growth factor (IGF), and EGF. All are necessary for

collagen formation. Epithelialization, angiogenesis, granulation tissue formation, and

collagen deposition are the principal steps in this anabolic portion of wound healing.9,10

3 | P a g e

1.2. Helicoll Regulatory:

Helicoll was approved by the FDA as a medical device on August 5, 2004 with 510(k)

number K040314. The product comes from USDA approved bovine sources with FDA

required regulatory documentation to maintain and monitor the safety and quality of the

procured animal derived raw materials.

1.3 Helicoll Manufacturing and Chemistry:

Helicoll is an acellular collagen matrix free of contaminants (the final production,

processing and packaging of Helicoll occurs in an FDA approved clean room).

Contaminants not eliminated during processing or packaging could cause an immunological

response when applied to the host wound which interferes with the healing process.7

Contamination from other types of collagen such as Type-II and Type-III are potentially

immunogenic and such types of collagen are completely removed in preparing Helicoll.

Our method was developed in order to address the problems presented by other commonly

used collagen preparations. Our EnColl process is predicated in part on the discovery that

collagen may be prepared in a manner in which all non-collagenous materials are removed,

while retaining the native molecular quaternary structure and other characteristic features of

collagen (e.g., length, diameter, and periodicity of collagen Type-I fibrils; see Figure 1).

Figure 1: Microphotographs of Helicoll collagen fibrils11

Image of meshwork of collagen fibrils. A: 100X; B: 1000X, showing periodic banding.

Helicoll is tested and manufactured in the FDA-certified clean room which is a controlled

environment that filters all incoming air to remove all dust particles and possible

contaminants that may interfere with the healing process. To be FDA certified, the clean

room must meet the standards for controlled environments set forth in ISO 14644-1.

Helicoll collagen dressing does not require refrigeration and can be stored at normal room

temperature for three years as stated in the FDA approval (Error! Reference source not

4 | P a g e

found.). NASA scientists in 2010, upon reviewing the product information and in

consideration possible use of the product on the 21-month Mars missions in 2035 and

beyond, determined that the product is ideal for their missions. They stated that they believe

the product has an incredible nine-year shelf life at standard temperature and pressure.

Important for their missions is ease of application, storage requirements, size and healing

rate.

The EnColl process may be used to prepare highly purified collagen from various animal

sources (including humans) as most, if not all, contaminating conjugated proteolipids and

phospholipids are removed through use of a specific mixture of organic solvents. Unlike

previously reported enzymatic methods and patents filed for collagen preparation,12

the

EnColl method utilizes a two-step enzyme treatment process. This two-step treatment

processes (“Twice Treatment Process” or “TTP”) renders collagen polymers non-

inflammatory when implanted.13

The use of papain, an enzyme extracted from papaya, is known to break the disulfide bonds

of cysteine14

. As many immunogenic molecules contain cysteine disulfide bonds15

, papain

may be used to degrade these molecules and render them non-immunogenic. In comparison

with other collagen preparations for biomedical applications, better results in terms of

reduced immunogenicity are obtained with EnColl’s collagen.13

In addition, papain has been reported to have a lytic effect on elastin, one of the contaminants

that is difficult to remove from purified collagen.12,13

Initial experiments involving a one-step

papain treatment to remove immunogenic sites from collagen were largely unsuccessful in

altering the in vivo performance of purified collagen. These observations led to the

development of the EnColl processes, which result in the breaking and loosening of the

natural crosslinks of collagen fibers (e.g., aldol condensation). In this manner, the papain

used in the second treatment step of EnColl’s patented process (i.e., papain is used in two

treatment steps) is provided access to most, if not all of the collagen molecules’ surfaces, and

facilitates the release of trapped immunogenic sites from the collagen preparation. These

developments resulted in one embodiment of the two-stage EnColl process, in which papain

is used at two specific stages of the process (i.e., before and after the treatment of the

collagen with a reducing and/or an unfolding agent). These methods therefore, provide means

to produce highly purified collagen that is non-immunogenic.16

The collagen is further bioactivated by varied degrees of controlled modification of

phosphorylation. Purified collagen can be chemically modified by covalently binding

phosphates to hydroxyl groups of hydroxylated amino acids. This reaction (an example for

serine is given below in

5 | P a g e

Figure 2) likely involves covalent bonding of phosphate to hydroxyl group of serine, tyrosine

and/or threonine, hydroxylysine and hydroxyproline.17

The reaction is controlled, in order to

limit the degree of reaction. EnColl's phosphorylated collagen renders unique abilities in the

growth of soft or hard tissue as needed by the physiological system.

Phosphorylation exposes multiple free binding sites which allow the collagen-connective

tissue framework to develop quickly. This proper alignment and binding of collagen fibres

causes the maturation process to accelerate wound healing. This allows epithelial

regeneration to occur leaving no scar formation.

Using patented technology, Helicoll collagen is phosphorylated to provide better

healing. Protein phosphorylation is reversible through protein phosphatases, enzymes that

hydrolytically remove specific phosphoryl groups from modified proteins. These

protein phosphatases are one mechanism for the termination of a signaling process.

Proteins undergo a huge number of post translational modifications. Only certain covalent

modifications such as acetylation, fatty acid acylation, glycosylation and phosphorylation are

reversible. Among these modifications, phosphorylation is an important and ubiquitous one.

The majority of the proteins involved in cell activation are subjected to reversible

phosphorylation. The sites of phosphorylation are serine, threonine and tyrosine hydroxyl

groups. Aspartic acid, histidine and lysine can also be phosphorylated. Phosphorylation of

tissue proteins is involved in natural cell differentiation of stem cells and in preventing

pathogenic bacterial invasion.

In nature, the phosphorylation of extra-cellular matrix protein is evidenced by the

accumulation of alkaline phosphatase in the regions of tissue formation or repair. The

significance of protein phosphorylation is to induce cell signal transduction through a cascade

of enzymatic reactions which are all documented in the literature. Collagen is the largest

native structural protein present at the sites of tissue repair remodeling or growth.

Phosphorylation of collagen makes the molecule biologically more active and becomes

essential for the cell signal transduction to happen.

Collagen has specific binding regions for all active components such as cell membrane

receptors, ligands, platelets, growth factors and other cytokines for proper interaction that can

result in repair, remodeling and regeneration of tissues. Phosphorylated collagen plays an

important role due to its ability to bring all necessary factors together and to activate them for

the desired result. Additionally, the phosphorylated collagen tends to attract divalent cations

such as Ca and Mg. Such divalent cations are essential for activating platelets and other

physiological events for faster wound repair or tissue growth. EnColl's patented and FDA

approved technology focuses on "collagen - phosphorylation" - and exploit the same for

extra-ordinary biomedical applications.

6 | P a g e

Figure 2: Serine phosphorylation

1.4. Biological Characteristics of Manufactured Collagen:

EnColl’s modified collagen has been shown to possess improved biological characteristics.

The modified collagen was found to have increased solubility features under neutral

conditions, which helps in the formulation of bioactive coatings on inactive surfaces.13

In one of the implant experiments, the modified collagen implants were analyzed for their

alkaline phosphatase (an enzyme involved in new tissue formation) activity. The assay

used15,18

was a calorimetric method using the measurement of o-carboxy-phenyl phosphate

(OCCP ) following the hydrolysis by alkaline phosphatase enzyme. Briefly, samples of

approximately 10 mg from each of the harvested collagen implants were dispersed at a rate of

1 mg in 1 mL of Tris buffer (0.1M Tris, pH 8.5) for five minutes. A small amount of

detergent (to a final concentration of 0.1M sodium deoxycholate) was added to the dispersed

samples to release of membrane-bound enzymes. The optical density was determined at 300

nm, at room temperature. The activity is expressed in units per mg of tissue, as based on the

number of micromoles of OCCP hydrolyzed per minute at 25°C, under the conditions

described above. The results showed significantly elevated amounts of alkaline phosphatase

(34% increase; p<0.0005) activity in the modified collagen implants as compared to the

unmodified implants.13

EnColl’s patented technology includes chemical modifications in solution or solid form of

collagen which can be used for a variety of purposes, including, but not limited to, biological

implants13,19-21

, grafts13,22

, transplants11,13

, and drug delivery13,23

.

A bioactive collagen dressing, such as Helicoll, induces platelet aggregation. Inflammatory

cells, neutrophils and macrophages invade the clotting area. After 4 days (refer to Figure 1) of

wound healing, there is a complete connective tissue bridge covering the wound. The site fills

with neutrophils and macrophages. At seven days, the inflammatory process recedes and the

repair process (proliferative phase) begins with the fibroblastic synthesis and deposition of

the extracellular matrix and collagen. Matured skin tissue develops consisting of bricks of

fibroblast cells that are mortared by the collagen produced by fibroblasts (see Figure 3). A

combination of cells and collagen provides a secure bridge over the interrupted skin tissue.24

7 | P a g e

Figure 3: H&E staining after Helicoll application

After Helicoll application the acute inflammatory cells, fibroblasts and blood vessels

proliferate into the collagen matrix. (50x). Absence of Lymphocytes indicates the non-

immunogenic property of the collagen in Helicoll.11

The role of pure bioactive Type I, non-immunogenic collagen, such as Helicoll, is to provide

binding and bridging sites for multiple chemokines (epidermal growth factor (EGF),

fibronectin, fibrinogen, histamine, platelet-derived growth factor (PDGF), serotonin, and von

Willebrand factor), necessary for building a connective tissue framework for epithelial

regeneration to occur.25-28

2. PRE-CLINICAL EXPERIENCE:

Numerous in vitro pre-clinical studies were conducted and are included in the Helicoll patent

5814328.13

The in vivo and in vitro tissue culture experiments using mice and rabbits demonstrated that

the delivery of growth factors was more effective when delivered through EnColl prepared

collagen as compared to native Type-I collagen.13

8 | P a g e

In Vivo Evaluation of Chemically Modified Collagen in adult New Zealand white rabbit

experiments showed more vascularization and fibroblastic in-growth in both of the

experimental groups (Example 6 and Example 2, see the Patent reference for further details).

Six of the rabbits (24 sites) were operated on bilaterally, with implants placed on both sides

of the dorsal mid-line. Each implant comprised 50 mg of dried collagen sample was rolled

into an approximately round ball and placed subcutaneously at each site. The animals were

observed for three weeks for gross indications of inflammation (e.g., redness, swelling, etc.).

No adverse responses were observed for any of the animals. After 3 weeks, the animals were

sacrificed and the implants were surgically removed and subjected to histology evaluations.

The control samples had relatively poor vascularization, as well as a prevalence of multi-

nucleated giant cells, reflecting the lesser biocompatibility of these samples.

2. CLINICAL EXPERIENCE WITH HELICOLL:

Figure 4: Clinical indications of subjects in Helicoll studies11

Clinical Situation Helicoll Used Patients Control

Patients

Donor Site 81 77

2nd Degree Burns 10 11

Diabetic Ulcers 6 5

Chronic Venous Ulcers 10 10

Contracture release & Bare

tendons, bones and joints

10 10

158 patients with split thickness skin grafts (STSG) were successfully treated with Helicoll in

2010. Study included measurement of pain reduction of patients treated with Helicoll

compared to control patients.11

Figure 5:

Healing rate of

superficial

burns treated

with Helicoll

or control11

13.1

6.2

0

2

4

6

8

10

12

14

16

Control (n = 11) HELICOLL (n = 10)

AV

ERA

GE

DA

YS

TO H

EAL

Healing Rate of Superficial Burns

9 | P a g e

Helicoll, in the clinical setting11

significantly reduced burn healing time, provided rapid pain

relief at the wound site, achieved 99.9% skin graft retention and reduced scarring, as well as

return of native skin color to the patient after several months. Helicoll also significantly

reduced the amount of hospital staff time required (dressing changes are less frequent as,

Helicoll can remain on wound for several days, wound inspection simplified as Helicoll is

semitransparent and wound can be assessed without removal of Helicoll), as well as total cost

of care by up to 50% over current therapies (product cost is up to 92 less expensive than some

competitors (Figure 10). Helicoll is available in large sizes so burns can be covered quickly.

Figure 6: Graft take following skin grafts in venous and diabetic ulcers

11

Helicoll is used for first and second degree burns, partial and full-thickness wounds, post

laser treatment, as well as pressure, venous, vascular, and diabetic ulcers. Trauma wounds

such as: Abrasions, lacerations, skin tears and donor sites are also indicated uses.

46.80%

96.70%

0%

20%

40%

60%

80%

100%

120%

Control, n=15(Skin Graft alone)

Experimental, n=16(Helicoll + Skin Graft on day 4 or 5)

PER

CEN

TAG

E O

F G

RA

FT T

AK

E

Percentage of Graft take at 4 weeks following Skin Graft Treatments on Venous and Diabetic Ulcers

10 | P a g e

Figure 7: Treatment of Post-Burn Contracture with Helicoll11

Helicoll can be placed on wounds caused by soft tissue necrosis secondary to radiation,

chemical burns or corrosives. The Helicoll is moistened with sterile water or normal saline

for six to ten minutes and placed in direct contact with the necrotic tissue. Daily dressing

changes are recommended with mechanical debridement of the necrotic tissue to reduce the

bioburden of the necrotic tissue and assist with autolysis.

Oxygen enhances the wound healing activity of collagen so Helicoll can be applied to

wounds that are undergoing treatment with hyperbaric oxygen.

It does not matter which surface of the Helicoll Wound Dressings is placed against the wound

surface. Helicoll must remain in contact with the wound by light pressure to ensure the

contact of the wound surface with the collagen to ensure proper healing.

Only areas with skin damage will interact with Helicoll. Any excess collagen (see Figure 8)

can be rinsed away with saline irrigation, so removal of the dressing does not interfere with

healing granulation tissue nor does it cause a painful experience for the patient. Helicoll is

also semi-translucent so that observation of the healing can be accomplished without

disturbing the healing tissue.

11 | P a g e

Figure 8: Photographs of wounds treated with Helicoll11

Figure 9: Photograph of wounds showing Helicoll incorporation11

12 | P a g e

2.1. Helicoll Advantages:

The usefulness of Helicoll over any other dressings in the market is well documented for the

treatment of diabetic ulcers, donor sites, burn treatments and other types of wounds. Refer to

Figure 4 for distribution of types of wounds treated in clinical studies.

With Helicoll, epithelialization occurs even in the inner areas of the wound site. This did not

happen with other collagen preparations used on the same wounds.7

As described in Section 1.3 of this document, native type-I collagen creates adhesion sites for

growth factors and also triggers “cell signal transduction” through which floating stem cells

convert into appropriate cell-lines to regenerate damaged tissue. Other collagen preparations

may not maintain the native chemistry of type-I collagen. The high purity type I collagen

dressing of Helicoll avoids any potential health risks normally caused by contaminating

immunogenic molecules like type-III, type-II collagens, elastin, glycosaminglycans, some

proteolipids, oligopeptides etc. Accordingly, the other dressings are cross-linked to minimize

the immunogenicity of contaminants at the expense of the needed bio-activity of collagen for

enhanced wound healing.

EnColl Corporation’s patented process uses a unique enzymatic process that result in a highly

purified collagen that is relatively non-immunogenic. It also renders a native un-crosslinked

collagen. Certain preparation methods of collagen products use crosslinking by chemicals

such as aldehyde without realizing that the resulting collagen is cross-linked and no longer

bioactive. If a collagen molecule is crosslinked, it loses the natural binding abilities to adhere

to cell surface receptors, growth factors, and other potential active molecules necessary for

the healing process to move forward. This impedes the natural cell-signaling properties of

collagen and thereby the crosslinked collagen reduces the wound healing capabilities of un-

crosslinked native collagen. If the collagen is native the cell-matrix interactions and the

bioactivity of cells will increase. Helicoll collagen provides this environment. It works to

reduce pain, scar formation and loss of pigmentation. Further it may also help to heal

wounds with limited blood supply in cases of arterial insufficiency.

Another advantage of Helicoll is that it has been shown to be safe for use on patients of all

ages from birth to centenarians. Helicoll provides hemostasis and accelerates tissue

remodeling and acts as an acellular dermal replacement product similar to Integra. Like the

Integra model, Helicoll promotes healing and neo-vascularization.

13 | P a g e

Some dressings are considered cytotoxic. Helicoll, however, has been shown to be extremely

bioactive, biocompatible and non-cytotoxic in vivo and in vitro.

Common benefits of Helicoll over other commercially available collagens in the market are:

Improved biocompatibility

Non-immunogenicity

Controlled bioresorbability

Cell attractability

Hemostatic ability

Structural stability

Target specificity

The disadvantages of using human skin allografts that do not apply to Helicoll include:

fear of HIV and other human infections

cross-linking or use of preservatives that can reduce the bioactivity of the graft

biohazardous material disposal concerns

immediate availability is quite difficult

limited shelf life

possible bacterial contamination

many eventually are rejected, making them a temporary rather than permanent wound

covering

14 | P a g e

Figure 10: Helicoll comparison with currently recognized skin substitutes

Helicoll collagen dressing normally comes in sizes from 2x2 inch to 15.75 x 15.75 inches. Larger sizes can be easily produced to cover large body areas.

To date, Helicoll has been used on over 77,000 patients (by May 2012) primarily by private

and university hospital professional health care providers. There have been no signs of

adverse reactions. We believe that the product Helicoll is the most effective (faster wound

healing), efficient (shorter time to apply and less dressing changes are required) , durable (has

high tensile strength) and easy to use (training physicians, nurses, medical assistants, patients

and care givers takes less than 15 minutes) wound-healing product on the market. It is safe

for neonates and infants or geriatrics and is currently used on wounds and burns. Helicoll

wound dressings are biocompatible and hypoallergenic.

HELICOLL™ COMPARISION WITH OTHER FDA APPROVED PRODUCTS PPRROODDUUCCTT HHEELLIICCOOLLLL™™ DDEERRMMAAGGRRAAFFTT®® AAPPLLIIGGRRAAFF®® OOAASSIISS™™ IINNTTEEGGRRAA™™

Matrix

Patented high purity

bovine Type-I

collagen, reconstituted

into sheets

Human fibroblast-

derived dermal

substitute on a

polyglactin mesh

Bi-layered

neonatal

fibroblast -

keratinocyte

(dermal -

epidermal) skin

substitute on

bovine Type I

collagen

Porcine

derived

acellular

small

intestinal

submucosa

(SIS)

Comprised of

collagen and

glycosaminoglycan

and a silicone layer

Size/shape

5x5 cm to 60x60 cm

in sizes with 0.2 mm

thickness

Rectangular, 5x7.5 cm

Circular, 8 cm

diameter, 0.75 mm

thickness

3x3.5 cm to

7x20 cm in

sizes

5x5 cm to 20x25 cm

in sizes

Sterilization Terminal sterilization Aseptically processed Aseptically

processed

Terminal

sterilization

Aseptically

processed

Shelf life 3 years at room

temperature

30 minutes at room

temperature; up to 6

months at -75°C

5 days at room

temperature

2 years at

room

temperature

1 year at room temp.

Handling

Rehydrates in saline

in 5 min.; easily

handled, sutured &

stapled

Shipped frozen;

elaborate prep and

appln procedures;

fragile

Shipped fresh on

an agarose

nutrient medium;

difficult to handle;

fragile

Rehydrates in

saline; easily

sutured &

stapled

Can be sutured &

stapled; easily

handled

Applications

to Heal 1-4 applications Up to 8 applications

Up to 5

applications variable variable

Approximate

cost per

application

US $100 per

application

US $500 per

application

US $1,200 per

application

US $800 per

application

US $1,100 per

application

Total

Advantages of

the product 7 of 7 0 of 7 0 of 7 2 of 7 1 of 7

15 | P a g e

Clinical evidence for product efficacy:

faster wound healing11

wound granulation and epithelialization in 4-5 days instead of 21–28 days (see

Figure 9)

reduced pain (see Figure 14)

lesser scar formation,

return of native skin pigmentation

Helicoll expedited healing in all cases and contained infection,

Promoted healthy granulation tissue, and stimulated a wound bed that better

supported a skin graft.

Itching was reduced.

Time to healing was hastened, and hence, total cost of treatment was also

lessened (see Figure 11, Figure 13)

3. CLINICAL TRIALS:

3.1. Use of Helicoll to treat skin ulcers:

3.1.1. 64 patients with ulcers were selected at random from different centers and treated

with varied acellular dermal replacement collagen dressings to compare the

effectiveness of Helicoll dressing with other collagen dressings. Healing was

visible as early as the 5th day after Helicoll treatment. There was no pain on

opening the dressing and patients had no discomfort. No adverse events were

reported.6

16 | P a g e

Figure 11: Rate of healing of wounds over 7 weeks in 64 patients with chronic ulcer

wounds using Helicoll6

3.1.2. 20 patients with ulcers were included to undergo treatment with Helicoll. In all

cases, wounds closed after a few bi-weekly and weekly applications. The wounds

remained closed for several months. It was noticed that epithelialization occurred

even in the inner areas of the wound sites, which did not occur when other

dressings were used.7

3.1.3. Helicoll was compared to traditional cotton gauze dressings for the management

of lower extremity ulcers in 18 patients. Although both study groups were

comparable at baseline, data indicate that the use of Helicoll resulted in faster re-

epithelialization.29

15

31

47

68

86

92 92

6 8

16

35

51

65 69

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7

Mea

n %

Ep

ith

eli

ali

zati

on

Time (Weeks)

Epithelialization Rate of Chronic Ulcers

Helicoll

Other CollagenDressing

17 | P a g e

Figure 12: Diabetic Foot Ulcer Treated with Helicoll25

The role of Helicoll collagens in foot care was demonstrated25

in independent

clinical studies showing at least 45% epithelialization of the foot ulcer wound

in 6 days. Further 30% healing improvement was observed with Helicoll

over other collagen products used for leg ulcer treatments.

3.2. Usage of Helicoll in treating Burns

3.2.1. Clinical study of 43 patients with second degree burns, age range 1 to 57 years

were randomized to receive Helicoll (n=23) or 1% silver sulphadiazine (n=22).

Helicoll resulted in a statistically significantly shorter time to healing (7.2 days

vs. 14.5 days, p=0.005). Healing was enhanced by 49.7% in the Helicoll group

compared to the silver sulphadiazine group. Itching was significantly decreased

in the Helicoll group (90.5% vs. 71.1% without itching).8,30

3.2.2. 26 burn patients were treated with Helicoll, compared to conventional dressings

in a multi-center study. There was a 4-fold increase in rate of healing in the

Helicoll group compared to the control gauze group.31

3.2.3. Vishal Mago, MD, unpublished report on “First & Second-Degree Burn

Treatment Trial of Collagen [Helicoll] Dressing vs. Silver Sulphadiazine Alone,”

as randomized, controlled study of efficacy and safety on 15 patients with

clinical burns, 2007. Better wound pain control with Helicoll.

3.3. Helicoll used to Heal Split Thickness Skin Grafts (STSG)

3.3.1. 60 patients with donor sites were selected at random at different centers and

treated with varied acellular dermal replacement collagen dressings to compare

the effectiveness of Helicoll with other collagen dressings. There was no pain on

18 | P a g e

opening the dressing and patients had no discomfort. Helicoll achieved a greater

patient comfort level as well as an accelerated healing rate compared to other

collagen dressings.6

Figure 13: Rate of epithelialization in 60 patients whose donor sites were treated with

Helicoll6

3.3.2. 22 patients with skin graft donor site wounds were included to undergo treatment

with Helicoll. Twenty of these patients had no pain, no restriction of mobility, no

infection when used per the protocol and the time to heal was significantly faster

when compared to other conventional dressings.7

3.3.3. 158 patients with STSG were successfully treated with Helicoll in 2010. Study

included measurement of pain reduction of patients treated with Helicoll

compared to control patients.

3.3.4. Reduced itching, increased range of motion, and overall increased patient

comfort were also experienced by patients treated with Helicoll for burns and

STSGs in this study (see Figure 14).11

Helicoll collagen dressing treatment showed 41% improvement over other

Standard Care Treatments in a Clinical Study of split skin graft donor sites.

Helicoll treated wounds healed in 7-10 days compared with 10-12 days with a

traditional treatment.11

Collagen reduces post-operative donor site pain. There was a significant

reduction in post-operative pain in the collagen dressings upon application of the

product, at days 1 and 2, and throughout the treatment process until complete

healing when compared to the other gauze groups (p < 0.02).11

Figure 14 below shows slight increase in pain on Helicoll patients on days 4 and

5 which corresponds to the infiltration of the live tissue cells into Helicoll as part

15

45

56

74

89 94 95

5 9

15

38

55

67 70

0

20

40

60

80

100

1 2 3 4 5 6 7

Me

an

% E

pith

elia

liza

tio

n

Time (Weeks)

Epithelialization rate of donor site wounds

Helicoll

19 | P a g e

of normal healing process.

Figure 14: Pain Response in Helicoll and Control Wounds11

Figure 15: Donor site treatment using Helicoll11

9.3 9.5 9.6 9.1

8.2 7.5

6.5 6.8

1.6 1.8 1.3

3.2 4.1

1.4 0.5 0.1

0

2

4

6

8

10

12

1 2 3 4 5 6 7 8

PA

IN

LEV

EL

Days after Treatment

PAIN RESPONSE FOLLOWING THE TREATMENT OF DONOR SITES

Control (n=77)

HELICOLL(n=81)

20 | P a g e

Figure 16: Collagen Patents

21 | P a g e

22 | P a g e

23 | P a g e

4. REFERENCES

Wild T, Rahbarnia A, Kellner M, Sobotka L, Eberlein T. Basics in nutrition and

wound healing. Nutrition. 2010;26(9):862-866.

King M. The Extracellular Matrix 2012;

http://themedicalbiochemistrypage.org/extracellularmatrix.php. Accessed July 2, 2012.

Wilner GDN, H.L.Lerocy, E.C. Activation of Hageman factor by collagen. The

Journal of Clinical Investigation. 1968;47(12):2608.

Rosenberg L. dlTJ. Wound Healing, Growth Factors. 2006; www.emedicine.com. Accessed

January 20, 2008.

Calne S, ed International consensus. Acellular matrices for the treatment of wounds.

An expert working group review. London: Wounds International; 2010.

Gunasekaran S KM, Dhanraj P. Bioactive Collagen Dressing for the Treatment of

Burns, Donor Sites, and Ulcers. World Biomaterials Congress Meeting. 2008.

Dhanraj P GS, DeWeese J, Sutkin H. How Native, Pure Type-I Collagen Dressing

Cures Ulcers Better Than Other Comparable Skin Substitutes. Association of Plastic

Surgeons of India. 2008.

Gunasekaran S KM, Dhanikachalam A, Narayan R. A comparative second-degree

burn treatment trial collagen dressing vs. silver sulphadiazine alone. American Society

for Dermatological Surgery. 2005.

Cho Lee A-R, Leem H, Lee J, Chan Park K. Reversal of silver sulfadiazine-impaired

wound healing by epidermal growth factor. Biomaterials. 2005;26(22):4670-4676.

Koempel JA, Gibson SE, O’Grady K, Toriumi DM. The effect of platelet-derived

growth factor on tracheal wound healing. International Journal of Pediatric

Otorhinolaryngology. 1998;46(1–2):1-8.

Dhanraj P MR, Herndon D. A Clinical Breakthrough in Wound Cover

"Bioengineered Collagen" - A Cost Effective and Expeditious Permanent Skin

Substitute. International Society for Burn Injuries. 2010.

Nimni ME. Collagen: Biochemistry, biomechanics, biotechnology. Vol 3. Boca

Raton, FL: CRC Press, Inc; 1988.

Gunasekaran S, Inventor. US Patent 5814328. Preparation of collagen using papain

and a reducing agent. 1998.

24 | P a g e

Smith EL, and J.R. Kimmel. Papain. In: P.D. Boyer HL, and K. Myrback, ed. The

Enzymes. Vol 4. 2 ed. New York: Academic Press, Inc.; 1960:138.

Brandenberger H. RH. Spectrophotometric determination of acid and alkaline

phosphatases. Helv. Chim. Acta. 1953;36:900-906.

Nimni ME, Cheung, D.T., Inventor. U.S. Patent 5374539. Process for purifying

collagen and generating bioprosthesis. 1994.

Gunasekaran S. Collagen/Phospholipid Interaction: Possible role in skin wound

dressing. Proc. Soc. Biomater. 1994;20:311.

Hofstee BHJ. Direct and continuous spectrophotometric assay of

phosphomonoesterases. Archives of Biochemistry and Biophysics. 1954;51(1):139-

146.

Simmons SJ, Jumblatt MM, Neufeld AH. Corneal epithelial wound closure in tissue

culture: An in vitro model of ocular irritancy. Toxicology and Applied Pharmacology.

1987;88(1):13-23.

Jumblatt MM, Neufeld AH. A tissue culture assay of corneal epithelial wound

closure. Investigative Ophthalmology & Visual Science. January 1, 1986

1986;27(1):8-13.

Kuwabara T, Perkins DG, Cogan DG. Sliding of the epithelium in experimental

corneal wounds. Investigative ophthalmology. 1976;15(1):4-14.

Coulson WF. The effect of proteolytic enzymes on the tensile strength of whole aorta

and isolated aortic elastin. Biochimica et biophysica acta. 1971;237(2):378-386.

Gunasekaran S, Inventor. US Patent 6127143. Preparation of purified and

biocompatible collagen using two proteolytic enzyme treatments and a reducing

agent. 2000.

Gunasekaran S, Inventor. US Patent 6548077. Purifying type I collagen using two

papain treatments and reducing and delipidation agents. 2003.

Gunasekaran S KM, Dhanikachalam A, Kilaru PG. Clinical review of a new bio-

engineered collagen dressing on diabetic ulcer wounds. Society for Biomaterials.

2007.

Nelson D.L. MC. Lehninger Principles of Biochemistry, 5th edition. 5 ed 2008.

Rodkey W, DeHaven K, Montgomery W, et al. Comparison of the collagen meniscus

25 | P a g e

implant with partial meniscectomy. A prospective randomized trial. Journal of Bone

and Joint Surgery; American volume. 2008;90(7):1413-1426.

Pentlow A, Smart NJ, Richards SK, Inward CD, Morgan JDT. The use of porcine

dermal collagen implants in assisting abdominal wall closure of pediatric renal

transplant recipients with donor size discrepancy. Pediatric Transplantation.

2008;12(1):20-23.

Rajendran MK ST, Sivakumar M, Gunasekaran S. Improved healing of lower-

extremity ulcers using advanced collagen membrane. Southern California Tissue

Engineering Symposium. 2002.

Gunasekaran S KM, Dhanikachalam A, Narayan R. A Comparative Second-Degree

Burn Treatment Trial of Collagen Dressing vs. Silver Sulphadiazine Alone. Society

for Biomaterials. 2006.

Gunasekaran S RM, Swaminathan T, Dhawan S. Modified Collagen for Burns

Enhances Healing Rate Possibly by Cell Signaling. 2003 American Society for

Dermatological Surgery Annual Meeting. 2003.

26 | P a g e

Attachment E - ASTM-F2212-2009 Collagen

Please refer to the attached Reference Document for the following Reference:

Standard Guide for Characterization of Type-I Collagen as Starting Material for

Surgical Implants and Substrates for Tissue Engineered Medical Products (TEMPs).

ASTM F 2212-09. Published Aug 2009.

27 | P a g e

Attachment F

Support Letters from a few of the potential end users

28 | P a g e

29 | P a g e

30 | P a g e

31 | P a g e

32 | P a g e

33 | P a g e